Prolonging the circulating half-lives of protein pharmaceuticals is of interest to patients and healthcare providers. Long acting protein therapeutics should require less frequent injections and can be effective at lower doses than proteins with shorter circulating half-lives. It is known that increasing the effective size of a protein can increase its circulating half-life by preventing removal of the protein by the kidney (Knauf et al., 1988; Mahmood, 1998). One method that can be used to increase the effective size of a protein is to use recombinant DNA technology to covalently fuse the protein of interest to a second protein. The larger fusion protein often has a longer circulating half-life than the non-fused protein (Capon et al., 1989; Zeng et al., 1995). One class of proteins that has been used frequently to create fusion proteins is immunoglobulins (Ig), which are major components of blood. Immunoglobulins occur in various classes known as IgG, IgM, IgA, IgD, and IgE (Roitt et al., 1989). Human IgGs can be further divided into various types known as IgG1, IgG2, IgG3 and IgG4, which are products of distinct genes. IgG1 is the most common immunoglobulin in serum (70% of total IgG) and has a serum half-life of 21 days (Capon et al., 1989; Roitt et al., 1989). Although less abundant, IgG4 also has a long circulating half-life of 21 days (Roitt et al., 1989)
Human IgGs have a multidomain structure, comprising two light chains disulfide-bonded to two heavy chains (referred to herein as a “tetrarner”; reviewed in Roitt et al., 1989). Each light chain and each heavy chain contains a variable region joined to a constant region. The variable regions are located at the N-terminal ends of the light and heavy chains. The heavy chain constant region is further divided into CH1, Hinge, CH2 and CH3 domains. The CH1, CH2 and CH3 domains are discreet domains that fold into a characteristic structure. The Hinge region is a region of considerable flexibility. Flexibility of the hinge can vary depending upon the IgG isotype (Oi et al., 1984; Dangl et al., 1988). IgG heavy chains normally form disulfide-linked dimers through cysteine residues located in the Hinge region. The various heavy chain domains are encoded by different exons in the IgG genes (Ellison et al., 1981; 1982).
Proteins have been fused to the heavy chain constant region of IgGs at the junction of the variable and constant regions (thus containing the CH1-Hinge-CH2-CH3 domains—referred to herein as the IgG-CH fusions) at the junction of the CH1 and Hinge domains (thus containing the Hinge-CH2-CH3 domains—referred to herein as IgG-Fc fusions), and at the C-terminus of the IgG heavy chain (referred to herein as IgG-C-terminal fusions).
IgG fusion proteins have been created most often with the extracellular domains of cell surface receptors (reviewed in Chamow and Ashkenaki, 1996). Examples of extracellular domains of cell surface receptors that have been joined using recombinant DNA technology to the CH or Fc domains of human or mouse IgGs include CD4 (Capon et al., 1989), tumor necrosis factor receptors (Mohler et al., 1993), CTLA4 (Linsley et al., 1991a), CD80 (Linsley et al., 1991b), and CD86 (Morton et al., 1996). Extracellular domains of receptors evolved to function when fused to other amino acids, i.e., the transmembrane and intracellular domains of the receptor, therefore it is not surprising that extracellular domains retain their ligand binding properties when fused to other protein domains such as IgG domains. Despite this, differences in ligand binding properties have been noted for certain extracellular domains. For example, a fusion protein comprised of the extracellular domain of CD4 to human IgG1-CH had 2-fold reduced affinity for the CD4 ligand gp120 than non-fused CD4 (Capon et al., 1989).
There are far fewer examples of proteins that are normally soluble, e.g., growth factors, cytokines and the like, which have been fused to IgG domains and retained full biological activity. Soluble proteins did not evolve to function when fused to other proteins and there is no reason to expect them to retain biological activity when fused to other proteins. In fact, in the majority of the published examples, biological activity of the fused cytokine/growth factor was significantly reduced relative to the non-fused cytokine/growth factor. Whether or not the cytokine/growth factor will function properly when fused to another protein will depend upon many factors, including whether the amino-terminus or carboxy-terminus of the cytokine/growth factor is exposed on the surface of the protein, whether these regions are important for biological activity of the cytokine/growth factor and whether the cytokine/growth factor is able to fold properly when fused to another protein. By their very nature, such factors will be highly protein-specific and unpredictable. Results with the few growth factor/cytokine fusion proteins that have been studied have shown how protein-specific biological activity of the fusion protein can be. In the majority of cases, biological activity of the fused growth factor/cytokine is severely reduced, whereas, in the minority of cases full biological activity of the growth factor/cytokine is retained. In one case where biological activity of the fusion protein was significantly reduced, modifying the amino acids at the junction between the cytokine/growth factor and the IgG domain resulted in a fusion protein with improved biological activity (Chen et al., 1994) This same modification did not improve biological activity of a second cytokine fused to the same IgG domain. [(Chen et al., 1994)
Growth factors that have been fused to IgGs include keratinocyte growth factor (KGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF-I). A KGF-mouse IgG1-Fc fusion protein was created by LaRochelle et al. (1995). On a molar basis, the fusion protein was 4-5-fold less active than KGF in stimulating proliferation of Balb/MK cells in an in vitro bioassay. The KGF-IgG-Fc fusion also had approximately 10-fold lower affinity for the KGF receptor on cells than did KGF. A fibroblast growth factor-human IgG-Fc fusion was constructed by Dikov et al. (1998). On a molar basis the FGF-IgG1-Fc fusion protein was approximately 3-fold less active than FGF in in vitro assays in stimulating DNA synthesis in NIH 3T3 cells. Shin and Morrison (1990) fused IGF-I to the C-terminus of IgG and found that the IGF-I-IgG C-terminal fusion protein had less than 1% of the in vitro biological activity IGF-I.
Examples of cytokines that have been fused to IgG domains include IL-2, IL-4, IL-10 and GM-CSF. Landolphi (1991; 1994) described an IL-2-IgG1-CH fusion protein, which included an extra serine between the C-terminus of IL-2 and the N-terminus of the IgG-CH domain. The IL-2-IgG1-CH fusion protein was purported to be as active, on a molar basis, in in vitro bioassays as IL-2, but no details were provided as to how protein concentrations were quantitated (Landolphi, 1991; 1994). Zeng et al. (1995) fused mouse IL-10 directly to the Fc region of mouse IgG2a; however the first amino acid of the Fc hinge region was changed from Glu to Asp. Zeng et al (1995) reported that the IL-10-IgG2a fusion protein was fully active in in vitro bioassays; however, only two concentrations of the fusion protein were studied, both of which were saturating. Given these high protein concentrations, only major differences (e.g., 100-fold) in bioactivities between the IL-10-mouse IgG2a fusion protein and IL-10 could have been detected. To detect smaller differences in bioactivities, one needs to analyze serial dilutions of the proteins in in vitro bioassays and calculate EC50s (the concentration of protein required for half-maximal stimulation) or IC50s (the concentration of protein required for half-maximal inhibition). EC50s of the IL-10-IgG2a and IL-10 were not reported by Meng et al. (1995). Chen et al. (1994) also constructed an IL-2-IgG fusion protein and reported that this fusion protein was fully active. Gillies et al. (1993) also reported creating a fully active IL-2 fusion protein comprising IL-2 fused to the C-terminus of an antiganglioside IgG antibody. Unexpectedly, Gillies et al. (1993) found that a fusion between the same antibody and GM-CSF displayed only 20% of wild type GM-CSF bioactivity. Chen et al. (1994) were able to create a fully active IgG-C-terminal-GM-CSF fusion protein by inserting four amino acids between the antibody molecule and GM-CSF. Unexpectedly, they reported that fusion of IL-4 to the same antibody using the same four amino acid linker resulted in an IL-4 protein with 25-fold reduced biological activity (Chen et al., 1994).
Qiu et al. (1998) described homodimeric erythropoietin (EPO) proteins in which two EPO proteins were fused together using flexible peptide linkers of 3-7 glycine residues. The peptide linker joined the C-terminus of one EPO protein to the N-terminus of the second EPO protein. In vitro bioactivities of the fusion proteins were significantly reduced (at least 4- to 10-fold) relative to wild type EPO (Qiu et al., 1998).
Thus, the literature indicates that the amino acids at the junction between the growth factor/cytokine domain and the IgG domain can have a profound influence on the biological activity of the fused growth factor/cytokine.
For use as human therapeutics, it is desirable that bioactivity of a growth factor/cytokine-IgG fusion protein be as close to wild type, i.e., the non-fused protein, as possible. Fusion proteins with high activity can produce greater therapeutic benefits at lower doses than fusion proteins with lower specific activities. This will reduce the cost of medicines and provide patients with greater therapeutic benefits. For use as a human therapeutic it also is advantageous that the protein be as homogeneous and pure as possible. Ig fusion proteins are synthesized as monomers, which can assemble to form disulfide-linked dimers and tetramers, depending upon the type of fusion protein. The relative proportions of monomers, dimers and tetramers can vary from lot to lot, depending upon manufacturing conditions. Lot to lot variability can result in heterogeneous product mixtures with varying activities. Because of their larger size, tetramers are expected to have longer circulating half-lives in the body than dimers or monomers, and dimers are expected to have longer circulating half-lives than monomers. For this reason, tetramers, dimers and monomers may produce different therapeutic effects in vivo. Purified, homogeneous preparations of tetramers, dimers or monomers are preferred as therapeutics because they will have defined specific activities. For use as human therapeutics it also may be beneficial to keep the length of any linker sequence joining the growth factor/cytokine domain to the Ig domain to a minimum to reduce the possibility of developing an immune response to the non-natural amino acids in the linker sequence. Joining the two proteins without an intervening linker sequence, referred to herein as a “direct fusion”, eliminates the possibility of developing an immune response to a non-natural linker and thus is one preferred form of an Ig fusion protein.
The literature on Ig fusion proteins merely provides examples for making “non-direct” cytokine/growth factor-Ig fusion protein, i.e., fusion proteins in which the growth factor/cytokine is joined to an Ig domain through a peptide linker. WO 99/02709 provides examples for joining EPO to an Ig domain by using a Bam HI restriction enzyme site to join DNA sequences encoding the proteins. This method can be used to create EPO-Ig fusion proteins containing linkers. Because Ban; HI has a specific DNA recognition sequence, GGATCC, which is not present at the junctions of the EPO or Ig-Fc, Ig-CH or light chain constant regions, this method is not useful for constructing EPO-Ig direct fusions.
EP 0464533A provides examples for making Ig fusion proteins that contain the specific three amino acid linker, AspProGlu, between the cytokine and Ig domains. The methods described in EP 0464533A rely on RNA splicing of separate exons encoding the cytokine and the Ig domain to create the fusion protein. The AspProGlu linker was required at the end of the cytokine/growth factor coding region because the RNA splicing reaction has specific sequence requirements at the splice site; the AspProGlu linker is encoded by nucleotides that may allow RNA splicing to occur. EP 0464533A provides an example for making an EPO-Ig fusion protein containing an AspProGlu linker using this method. The EPO coding region was modified to delete Arg 166 and add the amino acid linker, ProGlu, immediately following Asp165 of the EPO coding sequence. This resulted in the creation of an EPO-Ig fusion protein containing an AspProGlu linker (Asp165 of the EPO coding sequence provided the Asp of the linker sequence). The methods described by EP 0464533A are, therefore, not useful for creating Ig direct fusions or Ig fusions that do not contain AspProGlu or ProGlu linkers.
U.S. Pat. No. 5,349,053 describes methods for creating an IL-2-IgG-CH fusion protein that contains a Ser linker between the IL-2 and Ig domains. The methods described in U.S. Pat. No. 5,349,053 rely on RNA splicing of separate exons encoding the IL-2 and the IgG-CH domain. Because the RNA splicing reaction has specific sequence requirements at the splice site, the IL-2 coding region needed to be modified to add a TCA codon encoding serine to the carboxy terminus of the IL-2 coding region. The TCA codon was engineered to be followed by an intron splice junction sequence. This resulted in the creation of an IL-2-IgG-CH fusion protein containing a Ser linker. The methods described in U.S. Pat. No. 5,349,053 are not useful for creating cytokine/growth factor-Ig direct fusions, or cytokine/growth factor-Ig fusions that do not contain a Ser linker.
Growth factor/cytokine-IgG fusion proteins, including EPO-IgG and G-CSF-IgG fusion proteins, are contemplated in EP 0 464 533 A, WO99/02709 and Landolphi (1991; 1994). None of these references present any data regarding expression, purification and bioactivities of the contemplated EPO-IgG and G-CSF-IgG fusion proteins. Landolphi (1991; 1994) present bioactivity data for an IL-2-IgG1-CH fusion protein, but not for any other IgG fusion protein. The EPO-IgG1-Fc fusion protein contemplated by EP 0 464 533 A contains the two amino acid linker, ProGlu, between Asp 165 of the EPO coding sequence and the beginning of the IgG1-Fc hinge region. The carboxy-terminal amino acid in EN), Arg166 would be deleted. EP 0 464 533 A does not provide any information as to how the G-CSF coding region would be joined to the IgG1-Fc hinge region. Bioactivity data for neither protein is provided in EP 0 464 533. WO 99/02709 describes an EPO-mouse IgG2a-Fc fusion protein, but not an EPO-human IgG fusion protein. Bioactivity data for the EPO-mouse IgG2a-Fc fusion protein are not presented. WO 99/02709 also does not provide details as to the source of the EPO cDNA or human IgG genes used to construct the contemplated fusion proteins or the precise amino acids used to join EPO to the IgG domain. The reference cited in WO 99/02709 (Steurer et al., 1995) also does not provide this information.
WO 99/02709 postulates that a flexible peptide linker of 1-20 amino acids can be used to join EPO to an Ig domain; however the amino acids to be used to create the flexible linkers are not specified in WO 99/02709. Qiu et al. (1998) reported that EPO fusion proteins joined by flexible linkers of 3-7 glycine residues have significantly reduced biological activities (4-10-fold) relative to wild type EPO.
WO 99/02709 provides methods for using conditioned media from COS cells transfected with plasmids encoding hypothetical EPO-IgG fusion proteins to increase the hematocrit of a mouse. Such conditioned media will contain mixtures of monomeric and dimeric EPO-IgG fusion proteins as well as many other proteins. WO 99/02709 does not provide any evidence demonstrating that the methods taught are effective.
Thus, despite considerable effort, a need still exists for improved methods for creating growth factor/cytokine-Ig fusion proteins directly or with a variety of different linker types that preserve biological activity of the growth factor/cytokine domain of the Ig fusion protein. The present invention satisfies this need and provides related advantages.